A rapid and sensitive method for the determination of l -α-glycerophosphate in animal tissues

ANALYTICAL

A Rapid

BIOCHEMISTRY

3. 3y(i462 (1962)

and Sensitive Method L- a-Glycerophosphate EDWARD

From the Merck

Institute

for

for the in Animal

Determination Tissues1

of

I. CIACCIO

Therapeutic

Research,

Rahway,

New

Jersey

Received October 6, 1961 In the course of investigating the metabolism of n-a-glycerophosphate in normal and tumor tissues (l-3) a rapid, precise, and sensitive method for the determination of n-a-glycerophosphate has been developed and has been used in this laboratory for the last three years. This method utilizes the reaction: L-a-glycerophosphate

+ DPN -+ dihydroxyacetone

phosphate + DPNH + H’

catalyzed by n-n-glycerophosphate dehydrogenase and involves the spectrophotometric determination of DPNH. A method depending upon the measurement of the DPNH formed at equilibrium was suggested by Bublitz and Kennedy (4) and modified by Wieland and Suyter (5). Although recent modifications (6, 7) have improved the sensitivity of the original procedure, the long incubation periods needed, the possible instability of the enzyme under the conditions used (6, 8), and the extra handling involved, left room for improvement. The quantitative assay method described in this paper utilizes the rate of reaction rather than the estimation of the total DPNH formed at equilibrium. In addition to increasing the sensitivity from lo- to lOOO-fold over the previously published methods, the procedure is simplified to a rapid analytical method that requires no preincubation and thereby permits large numbers of determinations to be handled conveniently. As most enzymic assays involve the determination of the total product formed or substrate utilized at equilibrium rather than the reaction rate (9, lo), the analysis of substrates of other dehydrogenases by rate measurements is also discussed. The application of this method to the analysis of the L-cY-glycerophosphate content of a number of vertebrate tissues is described and the 1 This work was supported by the Cancer Chemotherapy National National Cancer Institute, under the NationaI Institutes of Health 43-pH-1886. 396

Service Center, Contract #SA-

L-wGLYCEROPHOSPHATE

DETERMINATION

data compared with those originally obtained (11) using a semimicro calorimetric method. MATERIALS

AND

397

by Leva and Rapoport

METHODS

Reagents Diphosphopyridine

dine nucleotide not in use.

Nucleotide.

(Sigma 99-100%)

A 0.1 M solution of P-diphosphopyriwas prepared and kept frozen when

Crystalline rabbit muscle a-glycerophosphate dehydrogenase (Boehringer-Calbiochem) was diluted to 0.5 mg/ml with distilled water; the diluted solution was stable in the frozen state for at least a month. wGlycerophosphate Sodium Salt (98oJo), Na&,H, (OH) ,PO,*6H,O (Eastern Chemical Co., Newark, N, J.). Polarimetric measurements (12) of this preparation indicated it was an equal mixture of D and L isomers. The content of L-Lu-glycerophosphate.6H,O was assumed to be 49%. Hydrazine-Buffer Solution, p1-I 9.8. A stock solution containing 1.1 M (5.5%) hydrazine hydrate (K 6s K Laboratories), 0.2 M (1.5%) glycine, and 0.02M MgCI, was adjusted to pH 9.8 with KOH. Other materials were obtained from commercial sources and used without further purification. Glycerophosphate

Dehydrogenase.

Instrumentation

Either a Cary recording spectrophotometer, Model 11, or a Beckman DU spectrophotometer connected to a recorder was used to measure the change in optical density at 340 rnp. The rate of reduction of DPNH during the first twenty seconds of the reaction was found to give the most precise and sensitive standard curve. Where a recorder is not available the total increase in optical density over the first twenty seconds of the reaction can be measured manually with little decrease in sensitivity or precision. In the system described, equilibrium was approached in approximately five minutes at room temperature (20-23’C). Procedure Into a cuvette of l-cm light path and 3-ml capacity were added 1.8 ml of the hydrazine buffer solution, 0.1 ml of DPN, 0.1 ml of cY-glycerophosphate dehydrogenase, and sufficient distilled water to give a total volume of 3.0 ml after consideration of the amount of standard or test solution to be added. The spectrophotometer was adjusted, after which the standard or test solution was rapidly added and stirred. This required three to five seconds. The optical density following the addition of subAnalytical

398

CIACCIO

strate was recorded. Standard curves were determined change in the initial twenty seconds of the tracing.

by measuring

the

Preparation of Test Samples

Tissues for analysis were removed quickly from anesthetized (Nembutal) animals and immediately frozen in a dry ice-ethanol mixture. This material while still in the frozen state was blotted dry, weighed, and quickly homogenized in a measured volume of cold 5% (0.3M) trichloroacetic acid, using 2 ml of acid per gram of tissue. Either a Waring Blendor or a Lourdes homogenizer gave satisfactory homogenates. The mixture was centrifuged at 2000 X g and neutralized, and the supernatant fluid was analyzed. In in vitro experiments involving the metabolism of L-a-glycerophosphate, the reaction was stopped at the appropriate time with 0.1 ml of 4.8M trichloroacetic acid per 3 ml of reaction mixture. After centrifuging, the supernatant fluid was analyzed. Neutralization was not required where 0.2 ml or less of sample was added per 3 ml of reaction mixture. RESULTS

Figure

1 illustrates

AND DISCUSSTON

the relationship

rnA moles/ml L-a-GLYCEROPHOSPHATE

between the amount

L-W

CONCENTRATION

1. Effect of increasing amounts of rrcr-glycerophosphate on rate _ formation. Standard AnaIysis System, optical density change: tnangies, initial rate (measured over 20 set); circles, total change at 20 sec. Gary Spectrophotometer Model 11; chart speed, I”/min with slide wire to optical density unit on full scale. FIG.

of

of DPNH tangent to

Recording give O-l.0

L-wGLYCEROPHOSPHATE

DETERMINATIOX

399

glycerophosphate in the reaction mixture and the rate of optical density increase. As there is a decrease in the rate even during the first twenty seconds, two graphs were constructed, one utilizing the initial rate as measured by a tangent drawn to the curve, and the other utilizing the increase in optical density over the first twenty seconds of the recording. The measurements of the total change during the first twenty seconds are proportional to the initial rates, as well as more convenient to measure, and give a linear standard curve from 2 to over 40 mpmoles L-a-glycerophosphate,/ml solution. The standard twenty-second curve, as shown in Fig. 1, has been reproduced with little variation in the course of many analyses in this laboratory. A precision of t5% was consistently obtained for the values on each standard curve. We have also determined standard curves after five minutes of incubation, at which time the production of DPNH approached equilibrium. At the same concentrations of a-glycerophosphate shown in Fig. 1, a standard curve measured at equilibrium is not reproducible from day to day and often does not go through the origin. This may be due to instability of the a-glycerophosphate dehydrogenase or of the end product DPNH (6, 8) or possibly to a drift in the spectrophotometer. At higher substrate concentrations where maximal sensitivity is not required, the equilibrium method will however give satisfactory results. The standard curve shown was confirmed by using known amounts of enzymically synthesized L-a-glycerophosphate. As there was good agreement between the synthesized LW and the racemic a-glycerophosphate (calculated as 49% ~-a), it is evident that the n-a-isomer is not interfering in the test system. In order to assure sufficient DPN in the analytical system the final concentration was kept in excess of 2 X 10e3M. This is necessary in view of the high Michaelis constant of a-glycerophosphate dehydrogenase with respect to DPN, of from 7 to 4 X 10w4M (6, 8). The hydraeine containing buffer at pH 9.8 serves two purposes: (1) dihydroxyacetone phosphate is trapped by hydrazine as well as destroyed by the high pH, thus pulling the reaction to the right, and (2) the equilibrium of the reaction at the high pH also favors the DPN reduction and concomitant glycerophosphate oxidation, while at a neutral pH glycerophosphate formation would be favored. The enzymic purity of the glycerophosphate dehydrogenase used in this assay was checked by substituting lactate, malate, propanediol phosphate, glycerol, succinate, fructose-1,6-diphosphate, ethanol, pyruvate, and glyceraldehyde-3-phosphate as substrate sources. No reduction of DPN was obtained with any of these compounds and, therefore, it was assumed that this method could be utilized for the quantitative analysis nf b-a-glpccrophosphate in tissue extracts,

400

cIAcc10

In order to test the possibility that inhibitory substances in the sample might interfere with the assay, tissues were analyzed with and without added quantities of L-a-glycerophosphate. Inhibitory substances have never been detected in vertebrate tissue extracts. It is possible that cinnamates, which are known constituents of plant tissues and are inhibitors of glycerophosphate dehydrogenase,2 would interfere in the assay of these materials. Table 1 illustrates typical recovery data obtained when L-a-glyceroTABLE RECOVERY

OF ADDED

1

L-a-GLYCEROPHOSPHATE~ Recovered

Amt. L-S-GP added (m~moles/ml)

from rat kidney

homogenate

mpmoles/ml

150.0 75.0 30.0 15.0 7.5

149.0 75.3 30.6 15.6 6.9

0 L-cr-Glycerophosphate was added to a 10% rat kidney homogenate. values were subtracted from figures given .

%

99 101 102 104 92 The endogenous

phosphate was added to a kidney homogenate before precipitation with trichloroacetic acid. The endogenous amount of L-a-glycerophosphate in the homogenate (16.1 m~moles/ml) was subtracted from the values shown. These results a.re typical of recovery studies routinely observed with other tissues. Bulbitz and Kennedy (4) found that trichloroacetic acid apparently agglutinated a-glycerophosphate to the precipitated protein and recommended metaphosphoric acid as a deproteinizing agent. We have never observed a loss in L-a-glycerophosphate recovery with the use of trichloroacetic acid; however, trichloroacetate is an inhibitor of a-glycerophosphate dehydrogenase2 at concentrations of 0.1 M or higher. Where such levels may be approached in the analysis system, the substitution of metaphosphoric acid is recommended. This method has been used to analyze a large number of normal and tumor tissues for n-a-glycerophosphate endogenously present (1) or produced by various metabolic reactions (2, 3). In Table 2 a few of the endogenous values obtained are compared to those reported by Leva and Rapoport (11) using a nonenzymic calorimetric method. A number of other substrates have been analyzed in this laboratory by means of enzymic rate reactions. In addition to L-a-glycerophosphate, * Ciaccio, E. I., unpublished

data

401 TABLE L-WGLYCEROPHOSPHATE

2

CONTENT

OF VARIOUS

~moles/gm Tissue

Rat Rat Rat Rat

anslyzed

Enaymic

1.5 0.6 0.3 0.5

liver (fed) liver (fasted) kidney brain

w-et weight

method0

Chemical

(0.3-0.85) (0.1-0.55) (0.36-0.75)

are of five to six determinations are given in parentheses. Leva and Rapoport (11).

methodb

1.4 0.7 0.4 0.4

(0.9-2.1)

a Averages each; ranges

b From

TISSUES

consisting

of pools

of two

to three

animals

dihydroxyacetone phosphate is conveniently determined by means of the oxidation of DPNH by a-glycerophosphate dehydrogenase in pH 7.5 triethanolamine buffer (13). A sensitivity limit of 2 mpmoles dihydroxyacetone phosphate/ml test solution has been obtained. Malic and lactic acids have been analyzed by methods identical to those described for L-a-glycerophosphate except that the appropriate crystalline enzymes, namely, malic and lactic dehydrogenases,” were substituted for the (Yglycerophosphate dehydrogenase. Similarly, pyruvate and oxalacetate have been determined by measuring the rate of oxidation of DPNH in triethanolamine buffer at pH 7.5. SUMMARY

A simple and rapid enzymic method has been described for the analysis of L-a-glycerophosphate which is sensitive down to 2 mpmoles L-a-glycerophosphate/ml solution. The use of measurements of the rates of reactions in place of determinations of the products at equilibrium in the analysis of L-a-glycerophosphate as well as other substrates shortens the time periods involved, requires less handling, increases the sensitivity many fold, and decreases the possibility of errors due to unstable enzymes, substrates, or products. REFERENCES 1. CMCCIO,

E.

Meeting, 2. CL~CCIO, 3. CIACCIO,

I.,

AND

Div. E. E.

ORANGE,

of Biological

I., AND KELLER, I., KELLER, D.

J. B., Abstracts of Bmerican Chemical Society Chem., September 13-18, 1960, p, 27C. D. L., Federation Proc. 19, 34 (1960). L., AND BOXER, G. E., Biochim. et Bio~hys. Acta

37, 191 (1960). 4. 5. 6.

BUBLITZ, WIELAND, BOLTVALIK,

’ Obtained

C.,

AND

O.,

KENNEDY,

SUYTER, J. J., AND NOLL,

from

AND

Worthington

E. P., J. Biol. Chem. M., Biochem. Z. 329, H., And. &o&em. Biochemical

211, 951 (1954). 320

(1957).

1, 269 (1960).

Corporation,

Freehold,

New

Jersey.

402

CiAcc20

7. HOHORST, H. J., KREUTZ, F. H., AND B~CHER, 8. YOUNG, H. L., AND PACE, N., Arch. Biochem. 9. DEVLIN, T. M., Anal. Chem. 31, 977 (1959).

TH., Biochem. Biophys. 75,

2. 332,

18

(1959).

125 (1958).

G. E., AND MCLEAN, P., Biochem. J. 61, 381 (1955). E., AND RAPOPORT, S., J. BioZ. Chem. 149, 47 (1943). Vol. II, p. 31. Wiley, New York, 1952. 12. BAER, E., in “Biochemical Preparations,” 13. BEISENHERZ, G., B&HER, TH., AND GARBADE, K. H., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. I, p. 391. Academic Press, New York, 1955. 10. GLOCK, 11. LEVA,